The polymerase chain reaction (PCR) is a technique for rapidly synthesizing a large number of copies of a defined segment of a nucleic acid molecule (see, for example, Mullis et al U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159). The PCR technique is extremely sensitive, theoretically requiring only a single nucleic acid molecule for amplification. PCR is an enzyme-mediated reaction typically incorporating a template molecule, a pair of oligonucleotide primers and a nucleic acid polymerase in a reaction medium comprised of the appropriate salts, metal cations, free nucleotides and pH buffering system. The PCR process is based on repeated cycles of denaturation of double stranded nucleic acid, followed by oligonucleotide primer annealing to the nucleic acid template, and primer extension by the polymerase. The oligonucleotide primers used in PCR are designed to anneal to opposite strands of the template molecule and are positioned to flank the target sequence to be amplified. As the primers are extended from their 3′ ends by the polymerase, the extension products provide copies of the original target sequence which can in turn act as template molecules for further rounds of amplification. PCR amplification results in the exponential increase of discrete nucleic acid molecules to obtain the desired amount of amplified nucleic acid product, the length of which is defined by the 5′ ends of the oligonucleotide primers.
While simple and specific in principle, PCR is prone to several types of unwanted artefacts that can be problematic to users and can compromise the selectivity and specificity of the technique. For example, non-specific amplification of fragments may result from one or both of the primers binding to a sequence(s) other than the target sequence, which can produce one or more fragments that are not the desired product. Non-specific binding of primers frequently occurs and can be due, for example, to alternate primer binding sequences being present in the target nucleotide sequence template, the presence of similar primer binding sequences on foreign contaminating molecules and/or sub-optimal primer sequence design. These non-specific nucleic acid products can be problematic especially when template nucleic acid containing the target sequence is present in few copies.
In some instances primers may bind to only partially complementary binding sites, and be extended even when a mismatch to the bound DNA is present at the 3′ end of a primer (e.g. see Kwok et al. 1990, “Effects of primer template mismatches on the polymerase chain-reaction—human-immunodeficiency-virus type-1 model studies.” Nucleic Acids Research 18(4): 999-1005).
Because of this, unwanted non-specific amplification can occur. When the strand of DNA resulting from mispriming is copied, a primer binding site is generated that fully matches the primer. Thus unwanted products can appear in a standard PCR that at both ends have binding sites that are completely complementary to the primers. Thus once this unwanted product is produced it is likely to be amplified with high efficiency. In other words, once mispriming has occurred, no further specificity is possible in a standard PCR. Although various types of probes can be used to specifically detect the wanted target, if too much non-specific amplification occurs, production of the desired product can be prevented or reduced to levels that are undetectable with the specific probe. This situation is most likely to occur in cases where the target of interest constitutes only a very small fraction of the total nucleic acid.
Non-specific amplification is also exacerbated when it occurs in the early rounds of the PCR cycle. Typically in existing PCR techniques, specificity due to the sequences of the primers is only achieved in early rounds of the PCR. Once a significant amount of product is generated, amplification proceeds at equal rates whether primers have extended from the intended sites flanking the target sequence or from non-specific amplicons arising from mispriming. That is, even where primers may have bound to and extension occurred from incorrect sites to which the primers have only partial complementarity, amplified non-specific products have the exact oligonucleotide primer binding sequence incorporated by virtue of the PCR technique, and these compete at equal efficiency with the nucleic acid template containing the target sequence for oligonucleotide primer binding in subsequent PCR cycles. In applications such as allele-specific PCR considerable care in primer design and considerable effort in technique optimisation are typically required to minimise mispriming.
In an attempt to limit the occurrence of non-specific product amplification, current PCR techniques may employ modified procedures aimed at increasing the specificity of primer binding to the target nucleic acid. This may involve, for example, altering the presence and concentration of different co-factors mediating the primer binding process or modifying the thermocycling of the PCR method. Alternatively, nested PCR may be utilized which involves two sets of primers used in two successive rounds of PCR. The first PCR amplification produces target and potentially non-specific products using the first set of primers, while the second PCR round uses new primers to amplify a secondary target within the first round target product in an effort to reduce the generation of non-specific product.
While existing PCR technique modifications improving primer binding specificity are useful, many require extensive trial and error which can be both time consuming and cumbersome. In addition, even after incorporating these techniques, non-specific products often remain which are inherently difficult to eliminate. Consequently, there is an ongoing need for improving PCR-based methodologies that allow for improved selective amplification of target nucleic acid sequences.
As with these methods, the new invention described here includes selectivity for sequences lying between the priming sites on the starting nucleotide sequence and can provide within a single reaction additional sequence specificity as is normally obtained using nested primers and two rounds of PCR.